WO2010118631A1 - Gène de protéine cristalline (cry56aa1) insecticide, sa protéine codée et ses utilisations - Google Patents

Gène de protéine cristalline (cry56aa1) insecticide, sa protéine codée et ses utilisations Download PDF

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Publication number
WO2010118631A1
WO2010118631A1 PCT/CN2010/000483 CN2010000483W WO2010118631A1 WO 2010118631 A1 WO2010118631 A1 WO 2010118631A1 CN 2010000483 W CN2010000483 W CN 2010000483W WO 2010118631 A1 WO2010118631 A1 WO 2010118631A1
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WIPO (PCT)
Prior art keywords
protein
gene
bacillus thuringiensis
expression vector
resistance
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PCT/CN2010/000483
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English (en)
Chinese (zh)
Inventor
李平
郑爱萍
朱军
王玲霞
王世全
邓其明
李双成
刘怀年
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四川农业大学
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Publication of WO2010118631A1 publication Critical patent/WO2010118631A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a novel Bt protein and its encoding gene and application. Background technique
  • Bacillus thuringiensis is a Gram-positive bacterium, which is widely distributed. It forms a companion crystal with insecticidal activity and is also known as insecticidal crystal. Insectididal crystal proteins (ICPs), which are encoded by the cr_y gene, are highly toxic to sensitive insects and non-toxic to higher animals and humans. In recent decades, Bt has been widely used in control A variety of pests such as Lepidoptera, Diptera, Coleoptera. In addition, Bt also has a controlling effect on a variety of pests such as Hymenoptera, Homoptera, Orthoptera, and Corydalis, and plant pathogenic nematodes, acarids, and protozoa. At present, Bt has become a powerful substitute for chemical synthetic pesticides in the control of farmland pests, forest pests and sanitary pests. Bt is also an important genetic source for transgenic insect-resistant engineering plants.
  • Bacillus thuringiensis subsp. israelensis subsp. israelensis (Bti) is a toxin-producing protein that has good insecticidal activity against mosquitoes and is widely used in the control of mosquitoes (Goldberg LJ, and Margalit J, 1977.
  • Cyt protein is cytosolic, has synergistic effect on certain Cry proteins and delays insect resistance (Wu, D., Johnson, JJ, and Federici, BA 1994. Synergism of mosquitocidal toxicity between CytA and CrylVD Proteins using inclusion Sproduced from cloned genes of Bacillus thuringiensis. Mol. Microbiol.
  • CytA enables CrylV endotoxins of Bacillus thuringiensis to overcome high levels of Cry IV resistance in the mosquito, Culex quinquefasciatus . Proc. Natl. Acad. Sci. 94: 10536-10540 )
  • Bacillus thuringiensis has been found for more than 100 years, and it has been widely used in the control of crop and horticultural plant pests, forest pests and sanitary pests, and has also achieved good results.
  • Bacillus thuringiensis due to the large-scale and repeated use of Bacillus thuringiensis, many insect populations have successively developed resistance to insecticidal crystal proteins to varying degrees.
  • the use of Bt insecticidal crystal protein-based insecticides has been used for more than 50 years. Initially, insect resistance to Bt has not been detected.
  • resistance problems have continued to exist in laboratories and It has been confirmed in field trials (M cG aU ghey, WH 1985.
  • a first object of the present invention is to provide a new BT virulence protein resource for the above deficiencies.
  • a second object of the invention is to provide a gene encoding the protein.
  • the virulence test on YWC 2 _8 showed that YWC2-8 has extremely high virulence to coleopteran pests, lepidopteran pests, diptera pests, and the like.
  • a pair of specific primers was designed according to the conserved sequence of 0>5 gene, and the genomic DNA was amplified. The results showed that the C ⁇ gene was present in the strain, and the full-length gene primer was further designed.
  • the Vy ⁇ ⁇ gene was cloned and its nucleotide sequence was sequenced. As shown in the SEQ ID No.l. list, the full length of the sequence SEQ ID N01 was 1992 bp, and the analysis showed that the GC content was 37.15%, encoding a protein consisting of 663 amino acids. The amino acid sequence thereof was determined as shown in SEQ ID No. 2.
  • the Bt protein of the present invention further comprises a protein derived from Cry56Aal having the amino acid sequence of SEQ ID No. 2 substituted, substituted and/or added with one or several amino acids and having an equivalent activity of Cry56Aal protein.
  • a gene of the invention includes a nucleic acid sequence encoding the protein.
  • codons suitable for expression of a particular species can be used, as desired, in view of the degeneracy of the codons and the preferences of the codons of the different species.
  • genes and proteins of the present invention can be cloned or isolated from strain YWC2-8, or obtained by DNA or peptide synthesis.
  • the gene of the present invention can be operably linked to an expression vector to obtain a recombinant expression vector capable of expressing the protein of the present invention, and the expression vector can be further transformed by a transgenic method such as Agrobacterium-mediated method, gene gun method, pollen tube pathway method or the like.
  • the host is introduced, and a transformant transformed with the Cry56Aal gene, such as a crop or a fruit tree, is obtained to have an insect resistance activity.
  • Figure 1 shows a 5 (1 ⁇ 27 full-length gene clone, M, marker; 1, cry56Aal gene.
  • Figure 2 shows the restriction endonuclease map of the recombinant plasmid pET-56Aa, wherein 1 recombinant plasmid pET-56Aa; 2, double digestion with pDE-30a with Nde l+EcoR I; 3, Nde l+EcoR I double digestion pET -56Aa; 4, inserted DNA; Ml, M2 are Marker.
  • Figure 3 shows SDS-PAGE detection of Cry56Aal in ⁇ :. coli BL21 (DE3), where M is the protein marker; 1. Negative control (E. coil BL21(DE3)(pET-30a)); Cleavage supernatant; 3. Cry56Aal inclusion body.
  • the present invention is a new strain of Bacillus thuringiensis isolated from the soil of Chengdu Plain, Sichuan province.
  • the strain has been in the General Microbiology Center of China Microbial Culture Collection Management Committee on January 12, 2Q09 (Address: Chaoyang District, Beijing) Datun Road No. 3, Institute of Microbiology, Chinese Academy of Sciences, 100101, China)
  • the classification is Bacillus thuringiensis, and the accession number is CGMCC.
  • the full-length sequence of Cry56Aal gene was cloned by the following method.
  • the total DNA of strain YWC2-8 was extracted using a genomic DNA purification kit (purchased from Thermos).
  • the primer sequences were designed as follows:
  • Thermal cycling reaction pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing temperature according to the primer, extension at 72 °C for 2 min, 30 cycles; extension at 72 °C for 5 min; stop reaction at 4 °C.
  • the amplification reaction product was electrophoresed on a 1% agarose gel, and the PCR amplification results were observed in a gel imaging system.
  • Fig. 1 a sequence of about 2000 bp was obtained by amplification, and the sequence was sequenced, and the nucleotide sequence thereof was as shown in SEQ ID No. 1, which was identical to the target sequence.
  • cry5ft4a gene and insecticidal activity assay A pair of specific primers cry56A was designed and synthesized based on the sequence of the open reading frame of the gene: 5'-GCGCATATG( ) ATGAGTATGAAATCATTGATTC -3'; cry56R: 5'-CGGAATTC (EcoR I) CACGTCAGGGGTAAATTCGATT -3', at the 5' end of the primers NI and EcoR I restriction sites.
  • the YWC2-8 plasmid was used as a template for amplification, and the amplified product was digested with Nde i and coR I. The digested product was ligated with the vector pET-30a(+) which was also digested and transformed.
  • / DH5a competent cells extracted by plasmid electrophoresis, verified that the size of the insert was in accordance with the intended purpose (Fig. 2), and then transferred to the recipient strain .co/.BL21 (DE3).
  • the recombinant plasmid was named pET-56Aa, and the recombinant containing the recombinant plasmid was named .co/i.BL21 (56Aa).
  • SDS-PAGE analysis showed that ⁇ y5(1 ⁇ 2 gene expression product was precipitated after sonication (Fig. 3), and the molecular weight was about 76kDa, which was consistent with the predicted protein molecular weight.
  • the gene expression products were respectively on beet armyworm, The results of bioassay of cotton bollworm, rice planthopper and Aedes aegypti showed that the expressed product had good insecticidal activity against all three insects.
  • the highest insecticidal activity against beet armyworm, LC 50 was 4.8 ng/mL;
  • the insecticidal activity of the planthopper was 6.7 ng/mL;
  • the LC 50 of Aedes aegyptus was 16.72 ng/mL; the insecticidal activity against cotton bollworm was the lowest, and the LC 50 was 23.16 ng/mL.
  • the protein of the present invention can be used for preparing Bt insecticides, and the genes can be converted into crops such as cotton, corn, rice, vegetables, etc., so as to have corresponding insect-resistant activities, thereby reducing the amount of pesticides used and reducing environmental pollution. Has important economic value and application prospects.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Pest Control & Pesticides (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Insects & Arthropods (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un gène de protéine cristalline Cry (Cry56Aa1) insecticide de la souche YWC2-8 de Bacillus thuringiensis (Bt), et sa protéine codée. Le gène ou son vecteur d'expression se révèle utile pour la préparation de plantes transgéniques et pour améliorer la résistance de plantes aux insectes. La protéine se révèle également utile pour la préparation d'insecticide à l'aide de la bactérie Bt.
PCT/CN2010/000483 2009-04-13 2010-04-13 Gène de protéine cristalline (cry56aa1) insecticide, sa protéine codée et ses utilisations WO2010118631A1 (fr)

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CN2009100823012A CN101531713B (zh) 2009-04-13 2009-04-13 Bt蛋白Cry56Aa1、其编码基因及应用
CN200910082301.2 2009-04-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438118A (zh) * 2022-02-17 2022-05-06 四川农业大学 在水稻、玉米中高效表达Bt蛋白Cry56Aa1抗草地贪夜蛾的方法

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* Cited by examiner, † Cited by third party
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CN101531713B (zh) * 2009-04-13 2011-01-12 四川农业大学 Bt蛋白Cry56Aa1、其编码基因及应用

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CN101531713A (zh) * 2009-04-13 2009-09-16 四川农业大学 Bt蛋白Cry56Aa1、其编码基因及应用
CN101531982A (zh) * 2009-04-13 2009-09-16 四川农业大学 苏云金芽孢杆菌ywc2-8及其应用

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CN101531713A (zh) * 2009-04-13 2009-09-16 四川农业大学 Bt蛋白Cry56Aa1、其编码基因及应用
CN101531982A (zh) * 2009-04-13 2009-09-16 四川农业大学 苏云金芽孢杆菌ywc2-8及其应用

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KUO, W S. ET AL.: "Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains on the Basis of Restriction Fragment Length Polymorphism of the PCR-Amplified DNA.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 62, no. 4, April 1996 (1996-04-01), pages 1369 - 1377 *
ZHU, J. ET AL.: "Characterization and expression of a novel holotype crystal protein gene, cry56Aal, from Bacillus thuringiensis strain Ywc2-8.", BIOTECHNOLOGY LETTERS., vol. 32, no. 2, February 2010 (2010-02-01), pages 283 - 288 *
ZHU, J. ET AL.: "Characterization of insecticidal crystal protein cry gene of Bacillus thuringiensis from soil of Sichuan basin and cloning of novel holotype cry gene", ACTA MICROBIOLOGICA SINICA, vol. 49, no. 3, 4 March 2009 (2009-03-04), pages 324 - 330 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438118A (zh) * 2022-02-17 2022-05-06 四川农业大学 在水稻、玉米中高效表达Bt蛋白Cry56Aa1抗草地贪夜蛾的方法

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